Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

Abstract

The methanogenic Archaea utilize a unique metabolic pathway for degradation of acetate under anaerobic conditions, and cleavage of acetate thereby accounts for a major proportion of the methane formed in the environment. The central reaction in this pathway is carried out by an unusual multienzyme complex, designated acetyl-CoA decarbonylase/synthase (ACDS), 1 which contains five different polypeptide subunits and accounts for as much as 25% of the soluble protein in species such as Methanosarcina thermophila and Methanosarcina barkeri growing on acetate. The ACDS complex catalyzes cleavage of the acetyl C-C bond using the substrates acetylCoA and tetrahydrosarcinapterin (H 4 SPt), a tetrahydrofolate analog which serves as methyl acceptor, and yields the products CoA, N 5 -methyltetrahydrosarcinapterin, CO 2 , and two reducing equivalents, as given in Reaction 1 (1).This overall reaction is made up of a series of partial reactions catalyzed by different protein subcomponents of the ACDS complex as shown in Scheme 1 (2). Acetyl-CoA binds to the ␤ subunit, and under low redox potential conditions, as required for activity, transfers the acetyl group to a nucleophilic center on the enzyme forming an acetyl-enzyme species and releasing CoA (Scheme 1, acetyl transfer) (2, 3). The acetyl intermediate then undergoes C-C bond cleavage by a reaction that is presumed to involve metal-based decarbonylation and/or methyl group migration (Scheme 1, cleavage). The nascent methyl group is then transferred to a corrinoid cofactor present on the ␥␦ subcomponent, which catalyzes subsequent methyl transfer to the substrate H 4 SPt (Scheme 1, methyl transfer) (2). The carbonyl group is oxidized to CO 2 by a process involving the ␣⑀ CO dehydrogenase subcomponent, with regeneration of the reduced form of the ␤ subunit. Previous studies on the ␤ subunit have focused on a C-terminally truncated form of the protein purified from the native ACDS complex following partial proteolytic digestion (2-4).The genes encoding the five ACDS subunits are arranged together in an operon along with one additional open reading frame in all species of Methanosarcina and in certain other methanogens as well (5-7, 9).2 The operon structure is shown in Scheme 2, with the designated genes and corresponding subunit molecular masses indicated for M. thermophila TM-1. The additional open reading frame encodes an accessory protein thought to be involved in nickel insertion, and nickel is present in both the large CO dehydrogenase subunit ␣ (CdhA) and in the ␤ subunit (CdhC) containing the active site for

Purification and Properties of the Klebsiella aerogenes UreE Metal-Binding Domain, a Functional Metallochaperone of Urease

“…The spectral changes of the control sample were somewhat more pronounced than what was observed in earlier studies (2) and led to the development of a defined peak at 366 nm. This Ni-induced absorbance is consistent with a weak thiolate-toNi 2ϩ charge transfer transition, as reported in other Ni-containing proteins (7,22) and model compounds (5), and suggests that Ni 2ϩ can coordinate to Cys79 when present at a high concentration. Maximal absorption of this chromophore required six equivalents of Ni 2ϩ per H144* dimer, suggesting that Ni binds to Cys79 only after the first three binding sites approach full occupancy.…”

A functional Ni-Ni-[4Fe-4S] cluster in the monomeric acetyl-CoA synthase from Carboxydothermus hydrogenoformans

“…Ni restored the acetyl-CoA͞CO exchange activities of ACS Mt ͞CODH Mt deprived of the metal by treatment with 1,10-phenanthroline (16,17) whereas Cu was inhibitory and not required for ACS activity (17). Ni also activated a recombinant apo ACS subunit from Methanosarcina thermophila, and other divalent metal ions could not substitute for Ni in yielding catalytic activity (18).…”

“…This signal was apparent from 10 to 130 K, with maximum intensity at 20 K. Integration of the signal revealed 0.14 mol of spin per mol of ACS Ch . A similar signal originating from the ACS͞CODH of other bacteria has been designated NiFeC signal (18,(35)(36)(37)(38).…”

“…Synthesis of acetyl-CoA was examined at 70°C by following acetyl-CoA formation from methylcobalamin, CO, and CoA, following published procedures (18,20,21). The assays were performed anaerobically in the dark in 2-ml vials.…”

AcsF Catalyzes the ATP-dependent Insertion of Nickel into the Ni,Ni-[4Fe4S] Cluster of Acetyl-CoA Synthase

“…ACS depends on a Ni,Ni-[4Fe4S] cluster (also called A-cluster) for activity, in which the two nickel ions have distinct coordinations: the nickel ion distal to the [4Fe4S] cluster (Ni d ) is coordinated by two amide nitrogen atoms and two cysteine thiolates within a Cys-X-Cys motif, holding nickel in a stable square-planar coordination environment (15,20,21). In contrast, the nickel ion proximal to the [4Fe4S] cluster (Ni p ) is weakly bound by three cysteine thiolates, is removable by 1,10-phenanthroline (22), and may be replaced by zinc and copper, thereby inactivating ACS (15,20,21,23,24). Ni p likely adopts different oxidation states during turnover and is the presumed place where substrates are activated and converted (13).…”